www.nature.com/scientificreports OPEN Raphidocelis subcapitata (=Pseudokirchneriella subcapitata) provides an insight into genome Received: 18 November 2017 Accepted: 8 May 2018 evolution and environmental Published: xx xx xxxx adaptations in the Sphaeropleales Shigekatsu Suzuki, Haruyo Yamaguchi , Nobuyoshi Nakajima & Masanobu Kawachi The Sphaeropleales are a dominant group of green algae, which contain species important to freshwater ecosystems and those that have potential applied usages. In particular, Raphidocelis subcapitata is widely used worldwide for bioassays in toxicological risk assessments. However, there are few comparative genome analyses of the Sphaeropleales. To reveal genome evolution in the Sphaeropleales based on well-resolved phylogenetic relationships, nuclear, mitochondrial, and plastid genomes were sequenced in this study. The plastid genome provides insights into the phylogenetic relationships of R. subcapitata, which is located in the most basal lineage of the four species in the family Selenastraceae. The mitochondrial genome shows dynamic evolutionary histories with intron expansion in the Selenastraceae. The 51.2 Mbp nuclear genome of R. subcapitata, encoding 13,383 protein-coding genes, is more compact than the genome of its closely related oil- rich species, Monoraphidium neglectum (Selenastraceae), Tetradesmus obliquus (Scenedesmaceae), and Chromochloris zofngiensis (Chromochloridaceae); however, the four species share most of their genes. The Sphaeropleales possess a large number of genes for glycerolipid metabolism and sugar assimilation, which suggests that this order is capable of both heterotrophic and mixotrophic lifestyles in nature. Comparison of transporter genes suggests that the Sphaeropleales can adapt to diferent natural environmental conditions, such as salinity and low metal concentrations. Chlorophyceae are genetically, morphologically, and ecologically diverse class of green algae1. Te group is dom- inant, particularly in freshwater, and plays important roles in global ecosystems2. Te Chlorophyceae are com- posed of fve taxonomic orders: Sphaeropleales, Chlamydomonadales, Chaetophorales, Chaetopeltidales, and Oedogoniales1. Te Sphaeropleales are a large group, and contain some of the most common freshwater species (e.g. Scenedesmus, Desmodesmus, Tetradesmus, and Raphidocelis)3,4, including some species used in applications such as bioassays and biofuel production. In particular, Raphidocelis subcapitata and Desmodesmus subspicatus are recommended for ecotoxicological bioassays by the Organization for Economic Cooperation and Development (OECD) (TG201, http://www.oecd.org/) because they have higher growth rates and greater sensitivity to various substances than other algae. Genome evolution in the Sphaeropleales is little understood compared to that of the Chlamydomonadales. In the Chlamydomonadales, genomes of four species (Chlamydomonas reinhardtii, C. eustigma, Gonium pec- torale, and Volvox carteri f. nagariensis) have been sequenced and analyzed thus far5–8. Comparative genome analyses have provided great insights into the evolution of green algae traits, such as fagella5, multicellularity6,7, and sexual reproduction9. In contrast, genome analyses of the Sphaeropleales are rare; only three genomes, that of Monoraphidium neglectum10, Tetradesmus obliquus11, and Chromochloris zofngiensis12, have been sequenced. Furthermore, their comparative analyses have not been performed. Te comparative analyses should provide insights into genome evolution of the Sphaeropleales and adaptation to diferent freshwater environments. M. neglectum contains large amounts of lipids under a wide range of pH and salt conditions, and thus shows potential Center for Environmental Biology and Ecosystem Studies, National Institute for Environmental Studies, Ibaraki, Japan. Correspondence and requests for materials should be addressed to S.S. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:8058 | DOI:10.1038/s41598-018-26331-6 1 www.nature.com/scientificreports/ for lipid production13. Its genome is 68 Mbp and encodes 16,761 proteins, including many genes related to car- bohydrate metabolism and fatty acid biosynthesis, and indicates that the vegetative cells have diploid characters. T. obliquus, which was formerly classifed as Acutodesmus obliquus or Scenedesmus obliquus, is a model organ- ism in the Sphaeropleales for biofuel production and organellar genetics. Its nuclear genome is 109 Mbp11, but its detailed genome structure (i.e. gene models and annotations) has not been described. Recently, the nuclear genome of C. zofngiensis (=Chlorella zofngiensis) has been sequenced; the genome size is 58 Mbp and it encodes 15,274 proteins. In the case of organellar genomes, the mitochondrial genome has unusual codon usages (i.e. UAG for leucine, not as a stop codon, and UCA as a stop codon, not for serine)14,15, and a split cox2 (cox2a, and cox2b)16. Te cox2b gene is located in the nuclear genome, and thus it appears to have been transferred to the nuclear genome via an endosymbiotic gene transfer (EGT)17. In the Chlamydomonadales, both cox2a and cox2b are in the nuclear genome; therefore, the genes of T. obliquus are thought to be an intermediate character in EGT18. Tese mitochondrial characters are conserved in the Sphaeropleales17,19. Te plastid genomes of the Sphaeropleales have fewer structural variations than the Chlamydomonadales and the OCC clade (Oedogoniales, Chaetopeltidales, and Chaetophorales)20. Terefore, the group is interesting for studying organellar and nuclear genome evolution in green algae. R. subcapitata NIES-35 was formerly known as Pseudokirchneriella subcapitata or ‘Selenastrum capri- cornutum’4. On the basis of 18S rRNA phylogeny, R. subcapitata belongs to the family Selenastraceae, order Sphaeropleales, but its phylogenetic position in the group has not been resolved4,21,22. Although well-resolved phylogenetic relationships have been recently reported for the Sphaeropleales, using plastid or mitochondrial genome-encoded proteins19,20,23,24, the organellar genomes of R. subcapitata have not been sequenced. In this study, we sequenced the nuclear, mitochondrial, and plastid genomes of R. subcapitata, and compared them to other Sphaeropleales species genomes to reveal genome evolution in the order based on well-resolved phyloge- netic relationships and to understand their genetic background in relation to high sensitivity to chemicals (e.g. metals). Te plastid and mitochondrial genomes provide insights into the phylogenetic relationships of R. subcap- itata and complex evolutionary histories in the order Sphaeropleales. Te nuclear genome of R. subcapitata is the most compact in this order, and comparison of proteins indicates that the Sphaeropleales can adapt to a variety of nutrient and environmental conditions. Results and Discussion Phylogenetic analyses. To infer the phylogenetic position of R. subcapitata within the Sphaeropleales, we performed phylogenetic analyses using two datasets: 55 plastid-encoded or 13 mitochondrion-encoded pro- teins (Fig. 1; Supplementary Fig. S1). In both trees, R. subcapitata formed a monophyletic group with the other Selenastraceae species, M. neglectum, Ourococcus multisporus, and Kirchneriella aperta. Tere was robust support (BP = 100 and BPP = 1.00) for inclusion of R. subcapitata in the Sphaeropleales; however, the topologies of the trees were diferent. Te tree using plastid-encoded proteins resolves the phylogenetic relationships better than the mitochondrial tree because it had higher supporting values (BPs at all nodes ≥70) and was based on more amino acids than that based on mitochondrion-encoded proteins. In the plastid-based tree, R. subcapitata was the most basal lineage in the Selenastraceae (BP = 94 and BPP = 1.00) (Fig. 1). M. neglectum and O. multisporus were sister species showing robust support (BP = 99 and BPP = 1.00). Te Selenastraceae was a sister group to T. obliquus, Neochloris aquatica, and Chlorotetraedron incus (BP = 99, BPP = 1.00). Chromochloris zofngiensis was monophyletic with the Selenastraceae, T. obliquus, N. aquatica, and C. incus, with moderate support (BP = 71, BPP = 0.9993). Terefore, C. zofngiensis was probably the most basal among the four species with available nuclear genomes of the Sphaeropleales. Evolution of plastid and mitochondrial genomes. In the Sphaeropleales, 17 mitochondrial and 11 plas- tid genomes have been submitted so far to comparative analyses10,15,19,20,23–26. We sequenced the complete mito- chondrial and plastid genomes of R. subcapitata and compared these sequences to those of other Sphaeropleales to reveal organellar genome evolution of the order, and particularly of the family Selenastraceae. Te mitochon- drial genome of R. subcapitata was circular and 44,268 bp in size (Supplementary Fig. S2a); this genome contained a large tandem repeat region with a 10-mer unit repeated at least 11 times at position 21,665. Protein-coding genes could be translated following the genetic code of the mitochondria of T. obliquus14. Tere were 13 con- served protein-coding genes (3 cytochrome oxygenases, 1 cytochrome b, 7 NADH dehydrogenase subunits, and 2 ATP synthase subunits), 6 fragmented rRNAs, and 28 tRNAs; as observed for other Sphaeropleales species, 16S rRNA and 23S rRNA were separated into two and four fragments, respectively (Supplementary Table S1). Cox2 was split and its N-terminus (Cox2a) was encoded in the mitochondrial genome, similar to other mito- chondrial genomes.